Hemophilia B, a hereditary recessive bleeding disorder, is successfully treated by replacement therapy consisting of the administration of preparations of human plasma derived (pdFIX) or recombinant coagulation factor IX (rFIX). The commercially available recombinant product, which is marketed under the trade name Benefix™, is manufactured by using stable transfected Chinese hamster ovary (CHO) cells co-expressing rFIX together with endopeptidase PACE/Furin, and is highly purified via multiple filtration and chromatographic steps (Kaufman et al., 1986; Wasley et al., 1993; Harrison et al., 1998). In clinical studies, Benefix™ has been shown to be safe and effective, but a 20 to 50% higher dosage than for pdFIX is needed for successful treatment. This is due to a 30 to 50% lower in vivo recovery for CHO derived rFIX than for pdFIX, as revealed by pharmacokinetic data collected from pre-clinical and clinical studies, where pdFIX and rFIX are compared in different animal models (Keith, Jr. et al., 1995; Brinkhous et al., 1996; Schaub et al., 1998; McCarthy et al., 2002), and clinical studies in hemophilia B patients (Keith, Jr. et al., 1995; White et al., 1997; White et al., 1998; Bjorkman et al., 2001; Roth et al., 2001; Ewenstein et al., 2002; Poon et al., 2002; Ragni et al., 2002; Kisker et al., 2003; Shapiro et al., 2005a). The circulating half-life of rFIX is not distinguishable from pdFIX preparations.
Biochemical comparison between pdFIX and CHO derived rFIX revealed differences in post-translational modifications (Bond et al., 1998). The lower degree of phosphorylation of a unique site at the activation-peptide amino acid serine 155 and the lower degree of sulfation of tyrosine 158 have been assigned to the lower in-vivo recovery of rFIX (White et al., 1997; Kaufman, 1998), although experimental evidence to proof this assumption has not been published to-date. These two modifications were identified to occur at less than 15% for the tyrosine-sulfation and at less than 1% for the serine phosphorylation in the recombinant protein, whereas the plasma derived protein has both modifications to more than 90% completed. Similar pharmacokinetic properties to Benefix™ were found for myotube-synthesized rFIX after adeno-associated viral vector mediated gene delivery in a mouse model (Arruda et al., 2001).
Therefore, a strong need exists for a new rFIX preparation which can be administered in a lower dosage than conventional rFIX preparation for a successful treatment.
Thus, it is an object of the present invention to provide a new rFIX preparation, wherein the rFIX has an improved in vivo recovery.